Targeted protein degradation has arisen as a powerful drug discovery paradigm for targeting undruggable proteins for proteasomal degradation. This technology utilizes bifunctional degraders called proteolysis targeting chimeras (PROTACs) that consist of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and proteasomally degrade proteins of interest. While targeted protein degradation can potentially be used to target any intracellular protein for degradation, a major challenge of this approach has been the dearth of E3 ligase recruiters. Using a chemoproteomic platform termed activity-based protein profiling (ABPP), which uses reactivity-based chemical probes to profile proteome-wide reactive, functional, and ligandable hotspots, we recently discovered that the anti-cancer natural product nimbolide, a triterpenoid isolated from Azadirachta indica or neem, covalently reacts with an N-terminal cysteine (C8) of the E3 ubiquitin ligase RNF114 in triple- negative breast cancer cells. Our preliminary data revealed that covalent modification of RNF114 by nimbolide leads to impaired ubiquitination of the tumor suppressor p21 through a nimbolide-dependent destabilization of the RNF114-substrate binding interaction, thus providing a potential mechanism for the anti-cancer effects of this natural product. This realization that nimbolide targeted a substrate recognition domain within RNF114 suggested that nimbolide could potentially be used as a novel recruiter for RNF114 for PROTAC applications. Consistent with this premise, we have shown that a PROTAC, XH2, linking nimbolide to a BRD4 inhibitor JQ1 led to proteasome-dependent degradation of BRD4 in breast cancer cells. This degradation was RNF114- dependent, as shown by BRD4 degradation in RNF114 wild-type cells, but not in RNF114 knockout cells. In this proposal, we will combine chemoproteomic and targeted protein degradation platforms to investigate the therapeutic potential of RNF114 modulators in cancer and exploit nimbolide and other covalent ligands targeting C8 of RNF114 as recruiters for PROTAC applications.